Semaphorins constitute a large family of more than 20 kinds of secretory and transmembrane proteins, and are classified into 8 classes (Cell 97, 551-552, 1999). Many of the semaphorins function as a chemorepellent or an attractant against nerves, and regulate the guidance of axons, nerve fiber fasciculation and branching, and synaps formation. (For overview, see Annu. Rev. Neurosci. 19, 341-377, 1996, Science 274, 1123-1133, 1996). In particular, it has shown that Class 3 secretory semaphorins have strong activities of growth cone collapse and against axons of dorsal root ganglion (DRG) neurons or sympathetic ganglion neurons. (Cell 75, 217-227, 1993, Cell 75, 1389-1399, 1993, Neuron 14, 263-274, 1995, Neuron 14, 949-959, 1995, Neuron 14, 941-948, 1995, Eur. J. Neurosci. 8, 1317-1321, 1996, Neuron 18, 193-207, 1997).
It has been shown that Neuropilins (Neuropilin-1 and Neuropilin-2) bind to Class 3 Semaphorins (Neuron 19, 547-559, 1997, Cell 90, 739-751, 1997, Cell 90, 753-762, 1997). It is also known that by inactivating Sema3A (previously, Semaphorin D, a member of Class 3 Semaphorin) gene (Neuron 19, 519-530, 1997) or Neuropilin-1 gene (Neuron 19, 995-1005, 1997) in the mouse by targeted gene disruption, the abnormal guidance of peripheral nervous fibers or the induction of dendrite formation were recognized, and Neuropilin-1 is necessary for transmitting chemical reactive signals induced in Sema3A.
On the other hand, Plexin is a membrane glycoprotein, that has originally been identified in Xenopus tadpole nervous tissue (Dev. Biol. 122, 90-100, 1987, Neuron 9, 151-161, 1992, Neuron 14, 1189-1199, 1995). Several kinds of Plexins have been identified in various animals, which have been classified into 4 subfamilies, that is, Plexin-A, -B, -C and -D (Cell 99, 71-80, 1999). In mice or human, at least 3 kinds of Plexin belonging to Plexin A subfamily, that is, Plexins A1, A2 and A3 have been identified [previously Plexins-1, -2 and -3 in the mice respectively (Biochem. Biophys. Res. Commun. 226, 396-402, 1996, Biochem Biophys. Res. Commun. 226, 524-529, 1996), and NOV, OCT and SEX in the human, respectively (Proc. Natl. Acad. Sci. USA 93 674-678, 1996)]. An ectodomain (extracellular domain) of Plexin A subfamily has repeats of three units of a cysteine cluster similar to cysteine-rich domain present in c-Met and Met-related receptor protein tyrosine kinases [they are called C1, C2 and C3 (Neuron 14, 1189-1199, 1995) or Met-related sequence (MRS; Proc. Natl. Acad. Sci. USA 93, 674-678, 1996) (for overview, see Proc. Natl. Acad. Sci. USA 93, 674-678, 1996; Dev. Neurosci. 19, 101-105, 1997). Further, the approximately 500 amino acids (aa) residues between the N-terminal of Plexin and the first cysteine cluster is significantly homologous to Sema domains shared by Semaphorin family (Cell 75, 217-227, 1993, Cell 75, 1389-1399, 1993, Trends Cell Biol. 6, 15-22, 1996, Eur. J. Neurosci. 8, 1317-1321, 1996).
Our previous studies using Xenopus has shown that Plexin is expressed in neurons constituting specific nervous domains such as the optic tecum (Dev. Biol. 122, 90-100, 1987), inner plexiform layer of the retina and photoreceptor cells (Neuron 9, 151-161, 1992) the olfactory system, the lateral neural circuit, and the auditory equilibrium system (Neuron 14, 1189-1199, 1995), and suggested that Plexins are involved in neuron cell contact (Neuron 14, 1189-1199, 1995), nervous fiber guidance and fasciculation (J. Neurosci. 15, 942-955, 1995), or organization of the inner plexiform layer of the retina (Neuron 9, 151-161, 1992). However, the molecular nature of Plexins and its role in the development of a nervous system are not understood well.
Some recent studies show that Plexins function as receptors for semaphorins. Plexin-C1 (VESPR), which has two cysteine clusters rather than three in an ectodomain is expressed in cells derived from the lymphatic system, and is used as a receptor for SemaVA, a Class V Semaphorin encoded and secreted by Poxvirus (A39R; Immunity 8, 473-382, 1998). A research on Drosophila (Cell 95, 903-916, 1998) show that Plexin A is used as a receptor transmitting chemorepulsive signals induced by Sema-1a, a Class 1 Semaphorin, and is shown that it regulates the nervous fiber fasciculation in CNS or motor neurons.
A recent study on the interaction between Plexins and Semaphorins (Cell 99, 71-80, 1999) elucidates that Sema4D, a Class 4 Semaphorin, binds to Plexin-B1, Sema7A, a Class 7 Semaphorin (GPI anchor-type) binds to Plexin C1. Further, members of Plexin-A subfamily (Plexin-A1 and Plexin-A3) form a complex with neuropilins (Neuropilin-1 and Neuropilin-2), and transmit Sema3A signals, a Class 3 Semaphorin, into cells (Cell 99, 59-69, 1999, Cell 99, 71-80, 1999). All these insights suggest a possibility that Plexins are receptor molecules crucial to transmit some semaphorin signals independent of Neuropilins. However, since Semaphorins have various structures and functions, receptors for Semaphorins and their the signal transmission mechanisms may vary, depending on the Semaphorin classes. At present, the receptors of Class 6 Semaphorins have not been identified yet, and their relationships with Plexins have not been elucidated.
The present invention is based on the insight that a receptor for Sema6C, a member of Class 6 Semaphorin, is Plexin-A1, and the object of the present invention is to provide screening methods for agonists or antagonists of Sema6C using Plexin-A1, and tools for the screening. Since Sema6C has the growth cone collapse activity against nerve cells and cell contraction activity, screening methods in the present invention are useful for selecting therapeutic agents or preventive agents for patients by promoting or inhibiting these activities.
The present inventors produced a transfectant expressing Plexin-A, and keenly screened a ligand which is a Plexin-A subfamily member using this. As a result of this, we found that a Class 6 transmembrane Semaphorin, a Sema6C (SemaY; Mol. Cell. Neurosci. 13, 9-23, 1999, SEQ ID NOS:8-10) specifically binds to Plexin-A1, and the recombinant proteins of the ectodomain (extracellular domain) of Sema6C induce contraction of fibroblasts or collapse of growth cones in the nerve cells, that express Plexin-A1, and we demonstrated that a receptor of a transmembrane semaphorin Sema6C is Plexin-A1. Accompanied by finding that a receptor of a Sema6C is Plexin-A1, it became possible to screen agonists or antagonists of Sema6C using Plexin-A1.
It is well known that Sema6C has a growth cone collapse activity (WO98/11216 pamphlet, Moll. Cell. Neurosci. 13, 9-23, 1999), and it is shown that the screening of antagonists of Sema6C is useful in developing promoting agents for neural regeneration (WO 98/11216 pamphlet). In the present invention, since a receptor for Sema6C is identified, it became possible to perform more effective and highly specific screening using Plexin-A1.
In particular, since Sema6C binding sites and Plexin-A1 are localized to the auditory system, and Sema6C has a growth cone collapse activity, the Sema6C is considered to be relevant to the suppression of auditory nerve elongation or the collapse of growth cone. The Sema6C is also considered to be relevant to the suppression of olfactory nerve elongation or the collapse of their growth cones. Therefore, the screening methods in the present invention is useful in screening for a therapeutic agents or preventive agents for various neural diseases, in particular auditory and/or olfactory nervous diseases. Since Sema6C also a cell contraction activity, the screening methods in the present invention can be used to screen agents that suppress or enhance cell migration using the contraction of cells expressing Plexin-A1 as an index. The methods can be used to screen therapeutic agents or preventive agents for the promotion of blood vessel formation migration of marignant cells.
Further, the present invention specifically has shown for the first time that Plexin-A1 is specifically expressed in the auditory neurons (all ganglia and nuclei constituting to auditory pathway). Therefore, the antisense strands of DNA/RNA encoding Plexin 1 or the antibodies against Plexin-A1 can be used for diagnosing auditory or olfactory nervous diseases.
Based on the insights mentioned above, the present invention has been completed.